Regulation of anchoring of the RIIalpha regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIalpha: implications for chromosome dynamics at mitosis.

CDK1 phosphorylates the A-kinase regulatory subunit RIIalpha on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localization of RIIalpha. Using an RIIalpha-deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RIIalpha or an RIIalpha(T54E) mutant, we show that RIIalpha associates with chromatin-bound A-kinase anchoring protein AKAP95 at mitosis and that this interaction involves phosphorylation of RIIalpha on T54. During interphase, both RIIalpha and RIIalpha(T54E) exhibit a centrosome-Golgi localization, whereas AKAP95 is intranuclear. At mitosis and in a mitotic extract, most RIIalpha, but not RIIalpha(T54E), co-fractionates with chromatin, onto which it associates with AKAP95. This correlates with T54 phosphorylation of RIIalpha. Disrupting AKAP95-RIIalpha anchoring or depleting RIIalpha from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstitution assay that mimics mitotic nuclear reformation, RIIalpha is threonine dephosphorylated and dissociates from AKAP95 prior to assembly of nuclear membranes. Lastly, the Reh cell line exhibits premature chromatin decondensation in vitro, which can be rescued by addition of wild-type RIIalpha or an RIIalpha(T54D) mutant, but not RIIalpha(T54E, A, L or V) mutants. Our results suggest that CDK1-mediated T54 phosphorylation of RIIalpha constitutes a molecular switch controlling anchoring of RIIalpha to chromatin-bound AKAP95, where the PKA-AKAP95 complex participates in remodeling chromatin during mitosis.

Mistargeting of B-type lamins at the end of mitosis: implications on cell survival and regulation of lamins A/C expression.

We previously showed that targeting of protein phosphatase 1 (PP1) to the nuclear envelope (NE) by the A-kinase anchoring protein, AKAP149, correlates with nuclear assembly of B-type lamins in vitro. We demonstrate here that failure of AKAP149-mediated assembly of B-type lamins into the nuclear lamina at the end of mitosis is followed by apoptosis, and induces expression of the gene encoding A-type lamins in cells that normally do not express lamins A/C. In HeLa cells, inhibition of PP1 association with the NE mediated by a peptide containing the PP1-binding domain of AKAP149 results in failure of B-type lamins to assemble, and in their rapid caspase-dependent proteolysis. However, assembly of lamins A/C is not affected. Nonetheless, apoptosis follows within hours of nuclear reformation after mitosis. In lymphoid KE37 cells, which do not express lamins A/C, inhibition of B-type lamin assembly triggers rapid synthesis and nuclear assembly of both lamins A and C before apoptosis takes place. The results indicate that nuclear assembly of B-type lamins is essential for cell survival. They also suggest that mistargeting of B-type lamins at the end of mitosis elicits a tentative rescue process to assemble a nuclear lamina in lymphoid cells that normally do not express lamins A/C.

HA95 is a protein of the chromatin and nuclear matrix regulating nuclear envelope dynamics.

We report a role for HA95, a nuclear protein with high homology to the nuclear A-kinase anchoring protein AKAP95, in the regulation of nuclear envelope-chromatin interactions. Biochemical and photobleaching data indicate that HA95 is tightly associated with chromatin and the nuclear matrix/lamina network in interphase, and bound to chromatin at mitosis. HA95 resides in a complex together with lamin B receptor (LBR), lamina-associated polypeptide (LAP)2 and emerin, integral proteins of the inner nuclear membrane. Cross-linking experiments, however, illustrate a tight association of HA95 with LBR and LAP2 only. Intra-nuclear blocking of HA95 with anti-HA95 antibodies abolishes nuclear breakdown in a mitotic HeLa cell extract. The antibodies inhibit nuclear membrane breakdown and chromatin condensation - the latter independently of nuclear membranes. However, lamina disassembly is not affected, as judged by immunological analyses of A/C- and B-type lamins. In contrast, immunoblocking of HA95 bound to condensed chromosomes does not impair chromatin decondensation, nuclear membrane reassembly or lamina reformation. Our results argue for a role for HA95 in anchoring nuclear membranes and lamins to chromatin in interphase, and in releasing membranes from chromatin at mitosis. The data also suggest that HA95 is not involved in initial binding of membranes to chromatin upon nuclear reassembly. We propose that HA95 is a central platform at the chromatin/nuclear matrix interface implicated in regulating nuclear envelope-chromatin interactions during the cell cycle.

Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly.

Subcellular targeting of cAMP-dependent protein kinase (protein kinase A [PKA]) and of type 1 protein phosphatase (PP1) is believed to enhance the specificity of these enzymes. We report that in addition to anchoring PKA, A-kinase anchoring protein AKAP149 recruits PP1 at the nuclear envelope (NE) upon somatic nuclear reformation in vitro, and that PP1 targeting to the NE is a prerequisite for assembly of B-type lamins. AKAP149 is an integral membrane protein of the endoplasmic reticulum/NE network. The PP1-binding domain of AKAP149 was identified as K(153)GVLF(157). PP1 binds immobilized AKAP149 in vitro and coprecipitates with AKAP149 from purified NE extracts. Affinity isolation of PP1 from solubilized NEs copurifies AKAP149. Upon reassembly of somatic nuclei in interphase extract, PP1 is targeted to the NE. Targeting is inhibited by a peptide containing the PP1-binding domain of AKAP149, abolished in nuclei assembled with membranes immunodepleted of AKAP149, and restored after reincorporation of AKAP149 into nuclear membranes. B-type lamins do not assemble into a lamina when NE targeting of PP1 is abolished, and is rescued upon recruitment of PP1 to the NE. We propose that kinase and phosphatase anchoring at the NE by AKAP149 plays in a role in modulating nuclear reassembly at the end of mitosis.

A kinase-anchoring protein (AKAP)95 recruits human chromosome-associated protein (hCAP)-D2/Eg7 for chromosome condensation in mitotic extract.

Association of the condensin multiprotein complex with chromatin is required for chromosome condensation at mitosis. What regulates condensin targeting to chromatin is largely unknown. We previously showed that the nuclear A kinase-anchoring protein, AKAP95, is implicated in chromosome condensation. We demonstrate here that AKAP95 acts as a targeting protein for human chromosome-associated protein (hCAP)-D2/Eg7, a component of the human condensin complex, to chromosomes. In HeLa cell mitotic extract, AKAP95 redistributes from the nuclear matrix to chromatin. When association of AKAP95 with chromatin is prevented, the chromatin does not condense. Condensation is rescued by a recombinant AKAP95 peptide containing the 306 COOH-terminal amino acids of AKAP95. Recombinant AKAP95 binds chromatin and elicits recruitment of Eg7 to chromosomes in a concentration-dependent manner. Amount of Eg7 recruited correlates with extent of chromosome condensation: resolution into distinct chromosomes is obtained only when near-endogenous levels of Eg7 are recruited. Eg7 and AKAP95 immunofluorescently colocalize to the central region of methanol-fixed metaphase chromosomes. GST pull-down data also suggest that AKAP95 recruits several condensin subunits. The results implicate AKAP95 as a receptor that assists condensin targeting to chromosomes.

Methyl tertiary butyl ether lacks tumor-promoting activity in N-nitrosodiethylamine-initiated B6C3F1 female mouse liver.

Methyl tertiary butyl ether (MTBE) is an additive in some formulations of unleaded gasoline (UG) that enhances octane and reduces carbon monoxide emissions from motor vehicles. MTBE in CD-1 mice and UG in B6C3F1 mice increased the incidence of liver tumors selectively in female mice in their chronic bioassays. Both agents were negative in in vitro tests of genotoxicity, and exhibit similar hepatic microsomal cytochrome P450 activity and hepatocyte proliferation after short-term exposure. We previously demonstrated that UG has hepatic tumor-promoting activity in DEN-initiated female B6C3F1 mice. Thus, we hypothesized that MTBE would have hepatic tumor-promoting activity in the same initiation-promotion model system in which UG was a hepatic tumor promoter. Twelve-day-old female B6C3F1 mice were initiated with a single i.p. injection of the mutagen N-nitrosodiethylamine (DEN) (5 mg DEN/kg, 7.1 ml/kg body weight) or saline. Beginning at 8 weeks of age, mice were exposed to 0 ppm or the hepatocarcinogenic dose of approximately 8000 ppm MTBE. After subchronic exposure, MTBE significantly increased liver weight and hepatic microsomal cytochrome P450 activity without hepatotoxicity or an increase in non-focal hepatocyte DNA synthesis. These are subchronic effects similar to those produced by UG. However, MTBE did not significantly increase the mean size of hepatic foci and volume fraction of the liver occupied by foci as compared to DEN-initiated controls at either 16 or 32 weeks. The lack of tumor-promoting ability of MTBE in DEN-initiated female mouse liver was unexpected and suggests that MTBE does not produce liver tumors through a tumor-promoting mechanism similar to that of UG.